Han-Chul Lee

Epigenetic regulation of microRNA-10b and targeting of oncogenic MAPRE1 in gastric cancer

MicroRNAs act as negative regulators of gene expression, and the altered expression of microRNAs by epigenetic mechanisms is strongly implicated in carcinogenesis. Here we report that the microRNA-10b gene (miR-10b) was silenced in gastric cancer cells by promoter methylation. In this study, using a methylation array and bisulfate pyrosequencing analysis, we found that miR-10b promoter CpGs were heavily methylated in gastric cancers. Clinicopathologic data showed that miR-10b methylation increased with patient age and occurred significantly more frequently in intestinal-type (28/44, 64%) than in diffuse-type (22/56, 39%) gastric cancers (P = 0.016). In addition, miR-10b methylation was also associated with an increase in expression of the oncogene that encodes microtubule-associated protein, RP/EB family, member 1 (MAPRE1; P = 0.004), which was identified as a potential miR-10b target. After 5-aza-2′-deoxycytidine treatment of gastric cancer cells, miR-10b methylation was significantly decreased, and expression of miR-10b and HOXD4, which is 1 kb downstream of miR-10b, was greatly restored. Moreover, decreased MAPRE1 expression coincided with increased miR-10b expression, suggesting that miR-10b targets MAPRE1 transcription. We also found that transfection with precursor miR-10b into gastric cancer cells dramatically decreased MAPRE1 mRNA and protein, resulting in a significant decrease in colony formation and cell growth rates. Thus, we show a tumor-suppressive role for miR-10b in gastric carcinogenesis. miR-10b methylation may be a useful molecular biomarker for assessing the risk of gastric cancer development, and modulation of miR-10b may represent a therapeutic approach for treating gastric cancer.

Genome-wide association of serum bilirubin levels in Korean population

A large-scale, genome-wide association study was performed to identify genetic variations influencing serum bilirubin levels using 8841 Korean individuals. Significant associations were observed at UGT1A1 (rs11891311, P = 4.78 x 10(-148)) and SLCO1B3 (rs2417940, P = 1.03 x 10(-17)), which are two previously identified loci. The two single-nucleotide polymorphisms (SNPs) were replicated (rs11891311, P = 3.18 x 10(-15)) or marginally significant (rs2417940, P = 8.56 x 10(-4)) in an independent cohort of 1096 individuals. In a conditional analysis adjusted for the top UGT1A1 variant (rs11891311), another variant in UGT1A1 (rs4148323, P = 1.22 x 10(-121)) remained significant; this suggests that in UGT1A1 at least two independent genetic variations influence the bilirubin levels in the Korean population. The protein coding variant rs4148323, which is monomorphic in European-derived populations, may be specifically associated with serum bilirubin levels in Asians (P = 2.56 x 10(-70)). The SLCO1B3 variant (rs2417940, P = 1.67 x 10(-18)) remained significant in a conditional analysis for the top UGT1A1 variant. Interestingly, there were significant differences in the associated variations of SLCO1B3 between Koreans and European-derived populations. While the variant rs2417940 at intron 7 of SLCO1B3 was more significantly associated in Koreans, variants rs17680137 (P = 0.584) and rs2117032 (P = 2.76 x 10(-5)), two of the top-ranked SNPs in European-derived populations, did not reach the genome-wide significance level. Also, variants in SLCO1B1 did not reach genome-wide significance in Koreans. Our result supports the idea that there are considerable ethnic differences in genetic association of bilirubin levels between Koreans and European-derived populations.

Genome-wide mRNA profiling and multiplex quantitative RT-PCR for forensic body fluid identification.

In forensic science, identifying a tissue where a forensic specimen was originated is one of the principal challenges. Messenger RNA (mRNA) profile clearly reveals tissue-specific gene expression patterns that many attempts have been made to use RNA for forensic tissue identification. To systematically investigate the body-fluid-specific expression of mRNAs and find novel mRNA markers for forensic body fluid identification, we performed DNA microarray experiment with 24 Korean body fluid samples. Shannon entropy and Q-values were calculated for each gene, and 137 body-fluid-specific candidate genes were selected. By applying more stringent criteria, we further selected 28 candidate genes and validated them by RT-PCR and qRT-PCR. As a result, we suggest a novel combination of four body-fluid-specific mRNA makers: PPBP for blood, FDCSP for saliva, MSMB for semen and MSLN for vaginal secretion. Multiplex qRT-PCR assay was designed using the four mRNA markers and DNA/RNA co-extraction method was tested for forensic use. This study will provide a thorough examination of body-fluid-specifically expressed mRNAs, which will enlarge the possibility of practical use of RNA for forensic purpose.

Identification and evaluation of age-correlated DNA methylation markers for forensic use

In forensics, age prediction is useful to narrow down the number of potential suspects because it can provide some general characteristics for predicting appearance. Previous genome-wide studies based on DNA methylation have reported age prediction algorithms using a penalized multivariate regression method known as elastic net and a few dozen to hundreds of CpG sites. Although more CpG sites may provide better accuracy than fewer CpG sites, this approach is not applicable to forensics because the amounts of crime-scene DNA are usually limited. In this study, we selected three age-correlated CpG sites, namely cg16867657 (ELOVL2), which is known to be an excellent age predictor, cg04208403 (ZNF423), and cg19283806 (CCDC102B), from HumanMethylation450 BeadChip datasets of 1415 individuals. Furthermore, we evaluated these markers in a 535-sample training set and a 230-sample validation set from Korean individuals using a pyrosequencing platform. From the training set, an age prediction model using the multiple linear regression method explained 91.44% of age-correlated variation in DNA methylation patterns. The standard error of estimate and mean absolute deviation were 6.320 and 3.156 years, respectively. In the validation set, the standard error of estimate and mean absolute deviation were estimated as 6.853 and 3.346 years, respectively. For the validation set, the model explained 91.08% of the variation in methylation and predicted age (±6years) with accuracy of 77.30% in the <60years age group and 57.30% in the older group (≥60 years). These results suggest that our three DNA methylation markers may be useful for age prediction in samples from Asian populations.

Microarray screening and qRT-PCR evaluation of microRNA markers for forensic body fluid identification.

MicroRNAs (miRNA) are a class of small (∼22 nucleotides) noncoding RNAs that regulate diverse biological processes at the post‐transcriptional level. MiRNAs have great potential for forensic body fluid identification because they are expressed in a tissue specific manner and are less prone to degradation. Previous studies reported several miRNAs as body fluid specific, but there are few overlaps among them. Here, we used a genome‐wide miRNA microarray containing over 1700 miRNAs to assay 20 body fluid samples and identify novel miRNAs useful for forensic body fluid identification. Based on Shannon Entropy and Q‐statistics, 203 miRNAs specifically expressed in each body fluid were first selected. Eight miRNAs were then selected as novel forensically relevant miRNA markers: miR‐484 and miR‐182 for blood, miR‐223 and miR‐145 for saliva, miR‐2392 and miR‐3197 for semen, and miR‐1260b and miR‐654–5p for vaginal secretions. When the eight selected miRNAs were evaluated in 40 additional body fluid samples by qRT‐PCR, they showed high sensitivity and specificity for the identification of the target body fluid. We suggest that the eight miRNAs may be candidates for developing an effective molecular assay for forensic body fluid identification.

Titin-cap (TCAP) polymorphisms associated with marbling score of beef

Marbling score (MS) is the major qualitative trait that affects carcass quality in beef cattle. In this study, we examined the association between genetic polymorphisms of the titin-cap gene (TCAP) and carcass traits in Korean native cattle (also known as Hanwoo). By direct DNA sequencing in 24 unrelated Korean cattle, we identified five sequence variants in 1.2kb of TCAP. Among them, four common polymorphic sites were selected for genotyping in the beef cattle (n=437). Pair-wise linkage analysis with four polymorphisms showed strong linkage disequilibrium (LD), and three major haplotypes (freq.>0.1) were constructed. Statistical analysis revealed that polymorphisms in intron1 (g.346G>A) and exon2 (g.592-597CTGCAG[Leu-Gln]insdel) showed significant association with marbling score (P(cor.)=0.003 and 0.02, respectively). One haplotype, ht2[C-G-G-del], also showed significant association with MS (P(cor.)=0.0004). Our findings suggest that polymorphisms in TCAP might be among the important genetic factors involved in carcass quality in beef cattle.

Identification of DNA methylation markers for lineage commitment of in vitro hepatogenesis

Hepatocytes that have differentiated from human embryonic stem cells (hESCs) have great potential for the treatment of liver disease as well as for drug testing. Moreover, in vitro hepatogenesis is a powerful model system for studying the molecular mechanisms underlying liver development. DNA methylation is an important epigenetic mechanism that influences differential gene expression during embryonic development. We profiled gene expression and DNA methylation of three cell states of in vitro hepatogenesis-hESC, definitive endoderm and hepatocyte-using microarray analysis. Among 525 state-specific expressed genes, 67 showed significant negative correlation between gene expression and DNA methylation. State-specific expression and methylation of target genes were validated by quantitative reverse transcription-polymerase chain reaction and pyrosequencing, respectively. To elucidate genome-scale methylation changes beyond the promoter, we also performed high-throughput sequencing of methylated DNA captured by the MBD2 protein. We found dynamic methylation changes in intergenic regions of the human genome during differentiation. This study provides valuable methylation markers for the lineage commitment of in vitro hepatogenesis and should help elucidate the molecular mechanisms underlying stem cell differentiation and liver development.

Forensic Body Fluid Identification by Analysis of Multiple RNA Markers Using NanoString Technology

RNA analysis has become a reliable method of body fluid identification for forensic use. Previously, we developed a combination of four multiplex quantitative PCR (qRT-PCR) probes to discriminate four different body fluids (blood, semen, saliva, and vaginal secretion). While those makers successfully identified most body fluid samples, there were some cases of false positive and negative identification. To improve the accuracy of the identification further, we tried to use multiple markers per body fluid and adopted the NanoString nCounter system instead of a multiplex qRT-PCR system. After measuring tens of RNA markers, we evaluated the accuracy of each marker for body fluid identification. For body fluids, such as blood and semen, each body fluid-specific marker was accurate enough for perfect identification. However, for saliva and vaginal secretion, no single marker was perfect. Thus, we designed a logistic regression model with multiple markers for saliva and vaginal secretion and achieved almost perfect identification. In conclusion, the NanoString nCounter is an efficient platform for measuring multiple RNA markers per body fluid and will be useful for forensic RNA analysis.

Serum cancer biomarker discovery through analysis of gene expression data sets across multiple tumor and normal tissues

The development of convenient serum bioassays for cancer screening, diagnosis, prognosis, and monitoring of treatment is one of top priorities in cancer research community. Although numerous biomarker candidates have been generated by applying high-throughput technologies such as transcriptomics, proteomics, and metabolomics, few of them have been successfully validated in the clinic. Better strategies to mine omics data for successful biomarker discovery are needed. Using a data set of 22,794 tumor and normal samples across 23 tissues, we systematically analyzed current problems and challenges of serum biomarker discovery from gene expression data. We first performed tissue specificity analysis to identify genes that are both tissue-specific and up-regulated in tumors compared to controls, but identified few novel candidates. Then, we designed a novel computation method, the multiple normal tissues corrected differential analysis (MNTDA), to identify genes that are expected to be significantly up-regulated even after their expressions in other normal tissues are considered, and, in a simulation study, showed that the multiple normal tissues corrected differential analysis outperformed the single tissue differential analysis combined with tissue specificity analysis. By applying the multiple normal tissues corrected differential analysis, we identified some genes as novel biomarker candidates. However, the number of potential candidates was disappointingly small, exemplifying the difficulty of finding serum cancer biomarkers. We discussed a few important points that should be considered during biomarker discovery from omics data.

Massive parallel sequencing of short tandem repeats in the Korean population

STR analysis using capillary electrophoresis has been the most widely used method for forensic DNA typing. Recently, massive parallel sequencing (MPS) technique has been emerging as an innovative tool to supplement or replace the conventional CE process. In this study, we evaluated the application of commercial MiSeqFGx™ forensic signature kit (Illumina Inc., San Diego, CA, USA) in the Korean population, including performance comparison with CE‐based STR profiling kits. The genotyping results of 209 unrelated random Korean individuals were summarized according to the International Society for Forensic Genetics guideline. The study revealed that 26 novel sequence variations in autosomal STR were newly found that had not been previously reported in other forensic literature. This indicates that MPS may be an effective supplementary tool for forensic DNA typing and the database to increase the discriminatory power of individual identification.